RNA viruses represent a significant portion of the high priority pathogens of concern to both human and animal health. The National Institute of Allergy and Infection Diseases' (NIAID) prioritized listing of human pathogens includes the RNA viruses: Hanta viruses, Dengue, Ebola, Marburg, Lassa, Hepatitis A and C, West Nile, and a large number of encephalitis viruses. The USDA prioritized list of infectious animal diseases include four RNA viruses within their top six ranked pathogens: Foot and Mouth Disease Virus, Rift Valley Fever Virus, common swine fever virus, and Japanese encephalitis virus. Viral outbreaks of these pathogens have significant direct impact on human health or dramatic indirect impact by their disruption of food supplies and related economic considerations. Hence, a rapid diagnostic would be an important and effective response to viral outbreaks. However, the current diagnostics are limited by both available technology and implementation.
For example, Dengue virus is a mosquito-borne viral pathogen prioritized by the World Health Organization (WHO) as it endangers 40% of the world's population, or 2.5 billion people, with over 50-100 million new infections each year. The WHO's ‘Gold Standard’ assays for Dengue include the plaque reduction neutralization assay, the haemmagglutination inhibition assay, and an IgM capture ELISA, and each of these methods require significant clinical infrastructure as well as both acute and convalescent samples from the patient for result validation. Current so-called “rapid” diagnostic tests have emerged based upon serology (IgM and IgG based methods), viral genetics (RT PCR methods), and viral isolation (cell culture and mosquito inoculation). However, these approaches are limited by costs, accuracy and sensitivity, and temporal considerations. Low-cost diagnosis of early onset Dengue is simply not practical, especially in remote settings where Dengue is rampant and clinical infrastructure is unavailable.
In the animal virus field, foot and mouth disease (FMD) is the most contagious transboundary animal disease affecting bovids and other cloven-hoofed animals. Significant economic losses result from its high morbidity, and from tourism and export trade restrictions imposed on affected countries. While the U.S. has not had a case of FMD since 1929, the country remains vigilant against its import and potential bioterrorism to protect against the devastating economic impacts of this disease. FMD is caused by infection of a picornavirus, a non-enveloped, positive-strand RNA virus. Previremic infection is often localized to epithelial tissues of the nasopharynx, due to aerosolized airborne contamination [Artz, 2010]. Active viral replication occurs during a preclinical phase of infection within this tissue and then continues in the lungs and endovascularly during the viremia phase. Viral replication is via an RNA-dependent RNA polymerase, D3pol, encoded by the virus whereby the RNA genome of the virus is replicated and packaged within the cytoplasm of infected cells.
Current methodologies for assay of FMD rely on detection of viral antigens and/or viral RNA, and do not require these markers to be active nor intact. Both approaches typically require laboratory clinical analyses. While fieldable kits are becoming available for ELISA-based detection, they have low sensitivity, and, importantly, do not provide an indication of active/transmissible infection. Charleston et al. (Science 2011) indicate that the infectious period of FMD is much shorter than previously realized (mean 1.7 days) and animals are not infectious until approximately 0.5 days after the onset of clinical symptoms. As such, costly remediation measures of herd culling may be unnecessary if accurate, fieldable methods for determination of infectiousness can be achieved.
The present invention overcomes the shortcomings of these assays and provides a highly fieldable, rapid diagnostic system for pathogenic RNA viruses